Cyclodextrin dimers with spacers having peptide structures for encapsulation of pharmaceutically active substances with potential high side-effects

ABSTRACT

A rigidly spaced, cyclodextrin dimers having a preselected breaking point within the spacer sequence so as to controllably release the active pharmaceutically active substance only after it reaches the desired treatment site is described. These preselected breaking points are stable in blood but are cleavable within cells. In preferred embodiments, the cyclodextrin-pharmaceutically active substance complex is targeted to specific sites via incorporation of specific antibodies for the targeted sites, typically by complexing a biotin-avidin system to specific antibodies which thereby targets the complex to a specific site. Once at the site as the complex is taken up into the cell the preselected break point is cleaved and the encapsulated pharmaceutically active substance becomes available for action within the targeted cell. This approach permits the use of highly effective and efficient pharmaceutically active substances, whose safety restricts use to last chance efforts or which are unable to qualify for human use due to their potential side effects. In a preferred embodiment peptide structures are used as part of the spacers between bridged cyclodextrins The cyclodextrin oligomers are complexed with pharmaceuticals with potential high side effects to safely, efficiently achieve the therapeutic action dsired.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to therapies with pharmaceuticallyactive substances with potentially high side-effects, which have to betransported in the blood and delivered to specific target cells withoutinfluencing healthy organs.

[0003] 2. Information Disclosure Statement

[0004] Therapies with very effective pharmaceutically active substancese.g. with antimitotic properties for cancer therapy often suffer fromthe fact that healthy organs are damaged severely by the therapy. Theseside effects often make the use of certain therapies impossible even ifthe therapy would be effective in curing the diseased organ. Thereforeit is desirable to achieve specific targeting of the pharmaceuticallyactive substances to the diseased organ or cells, and moreover toinhibit the delivery of the pharmaceutically active substance to healthyorgans. Often a localized application of the pharmaceutically activesubstance to the diseased site is not possible, but the pharmaceuticallyactive substance has to be administered systemically through the bloodstream and is accessible for the whole organism. One way to prevent thisis to build complexes with carrier molecules that mediate an inerttransport through the blood and selectively release the pharmaceuticallyactive substance at the target site.

[0005] In patent description in Pat. Ser. No. 09/554,223 by Jörg G.Moser (published as WO 9924474) a method was introduced to detoxifypharmaceuticals with dangerous side effects by physical encapsulationinto cyclodextrin [CD] oligomers with a relatively rigid spacerstructure B.

[0006] Cyclodextrins are annular glucose polymers, which are calledalpha, beta or gamma cyclodextrin depending on the number of glucosemoieties present, namely for 6, 7 or 8, respectively. A lipophiliccavity exists in the center of a cyclodextrin, where lipophilicsubstances can be enclosed. This property of cyclodextrins can be usedto render hydrophobic substances water soluble. Preferably cyclodextrinoligomers are able to encapsulate hydrophobic substances. The bridgingstructures or spacers between the cyclodextrins determine the distancebetween the cavities and thereby the size of the molecule that can beencapsulated. The spacer structures have to be rigid to ensure thecorrect orientation of the cyclodextrin moieties for the retention ofthe cavity structure. Therefore the spacer structures contain preferablychemical bonds that cannot rotate freely.

[0007] The protected molecule is released upon cleavage of either thecyclodextrins or the bridging structures between the cyclodextrins. Thenecessary destruction of the complex and the consequent liberation ofthe included pharmaceutically active substance at the target site can beeffected easily by hydrolysis of the cyclodextrin by a specific enzyme(Moser Ser. '223) or preferably by destruction of the spacer B′. In bothcases the affinity between pharmaceutically active substance andcovering CD's ceases by 4 orders of magnitude, and the pharmaceuticallyactive substance slips out of the complex into the next living cell. Thesynthesis of CD-dimers is well known (See, for example A. Rübner et al.J. Inclin. Phenom. 27 69-84 (1997)). Antimitotics like taxanes can beencapsulated this way.

[0008] The targeting of the complex to the diseased organ, e.g. a tumor,can be mediated by specific antibodies, preferably by using thebiotin-avidin (bAV) system. In particular, it has been tried to couplecomplexes from CD-dimers and pharmaceuticals with biotin-avidin (bAV)systems in order to connect the complex with biotinylated monoclonalantibodies (b-mAB's) and, thereby, to concentrate the complexspecifically to xeno-transplanted tumours in nude mice. In theseexperiments, the effect was not statistically significant even thoughthe administered concentrations were very high.

[0009] Therefore, it is object of the present invention to improve theeffect of complexed pharmaceutically active substances by changing thestructure of the spacer of the CD dimer in a way that the complex can beconcentrated at the desired site and that the pharmaceutically activesubstance is released intracellularly where its action is mostlyefficient. Moreover, inert transport of the pharmaceutically activesubstance through the blood is possible, contrary to the prior art.

BRIEF SUMMARY OF OBJECTIVES OF THE INVENTION

[0010] It is an object of the invention to provide cyclodextrin endcapped structures connected by rigid spacer sequence to encapsulatepharmaceutically active substances, where the spacer sequence has apreselected breaking point, which is stable in blood but cleavablewithin cells.

[0011] It is another object of the present invention to use, as spacers,peptide structures that are not cleavable by the proteolytic enzymes inblood but are sensitive to intracellular enzymes.

[0012] It is a further object of the present invention to modify thepeptide spacer with biotin residues in different positions to couple thecomplex via a biotin avidin system to specific antibodies and therebytarget the complex to specific sites.

[0013] Briefly stated, the present invention provides a Rigidly spaced,cyclodextrin dimers having a preselected breaking point within thespacer sequence so as to controllably release the activepharmaceutically active substance only after it reaches the desiredtreatment site. These preselected breaking points are stable in bloodbut are cleavable within cells. In preferred embodiments, thecyclodextrin-pharmaceutically active substance complex is targeted tospecific sites via incorporation of specific antibodies for the targetedsites, typically by complexing a biotin-avidin system to specificantibodies which thereby targets the complex to a specific site. Once atthe site as the complex is taken up into the cell the preselected breakpoint is cleaved within the cell and the encapsulated pharmaceuticallyactive substance becomes available for action within the targeted cell.This approach permits the use of highly effective and efficientpharmaceutically active substances, whose safety restricts use to lastchance efforts or which are unable to qualify for human use due to theirpotential side effects. In a preferred embodiment peptide structures areused as part of the spacers between bridged cyclodextrins Thecyclodextrin oligomers are complexed with pharmaceuticals with potentialhigh side effects to safely, efficiently achieve the therapeutic actiondesired.

[0014] The above and other objects, features and advantages of thepresent invention will become apparent from the following detaileddescription.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0015] The essential element of the present invention is the provisionof a preselected breaking point, which is stable in blood but cleavablewithin specific cells, in a system capable of transporting hydrophobicmoieties through the blood stream. Once a targeted cell site is reached,the carrier system cleaves to provide access of the hydrophobicpharmaceutically active substance moiety in the cell to initiatetherapeutic action as required by the medical treatment. Since the bloodcontains several proteolytic enzymes the breaking point is selected toonly be sensitive to intracellular enzymes. The structure of the carriersystem is preferably a cyclodextrin (CD) dimer with a rigid spacerseparating the CD ends, wherein the length and other properties of thespacer are selected to allow capture and retention of a pharmaceuticallyactive substance within the cavity formed between the ends. Thepreselected breaking point preferably is in the spacer structure alongwith moieties which may target the desired cell sites, where therapeuticaction is required.

[0016] When a peptide structure is used as a spacer structure withbreaking point, free lysine has to be avoided due to the frequenttrypsin-like specificity of blood proteases. Intracellular proteases ofthe proteasomes contain chymotrypsin. A compatible sequence for thespacer is e.g.:

[0017] The preselected breaking point is labelled by the arrow (↑).Sequence (I) would have a length between the CD moieties of 6.5 A. Thesequence allows connection to a b-mAB system over the avidin orstreptavidin. The basic CD, as a 6-biotinylated derivative, is known.Within the spacer sequence, every further amide bond would elongate thespacer length by 2.7 Å, every additional —CH₂-group by 1.5 Å.

[0018] Therefore, for guest molecules with a greater Van derWaals-distance more amino acids and/or methylene groups (at the CDmoieties) can be inserted into the spacer structure. For a distance of8.5-9.0 Å the following sequence is preferred:

[0019] Minor changes in length are effected by exchange of the propyl-to butyl residue [IIa]. For more extended guest molecules a repeatedbiotinylation inside the spacer is possible, e.g. an adjacent peptide:

[0020] This complex allows for polyphasic accumulation of a complexedpharmaceutically active substance, e.g. Paclitaxel, at the target site,as just noted Moser Ser. No. 09/554,223, but now the added break pointbetween Tyr-Asp provides easier access to the encapsulatedpharmaceutically active substance. The position of theLys(biotinyl)-residues is variable with respect to the breaking point.The following structure (IV) would be equivalent or better as (III):

[0021] As control preparations for in vitro experiments the followingsequences are suitable:

[0022] (II′) CD-Tyr-Pro-Lys(biotinyl)-aminopropylamido-CD

[0023] which cannot be split i.e. does not have a preselected breakpoint; and

[0024] (II′) CD-Tyr-Pro-Lys(Me)-aminopropylamido-CD

[0025] which can neither be split nor can it be attached to a targetingmoiety. `

[0026] All peptide structures described are found only in proteins withMr 10exp5 Da (EMBL Data Service), but are uncommon enough to be detectedby the ubiquitin system during receptor mediated uptake and so, aresplit by enzyme activities of the proteasomes. This splitting at thepreselected breakpoint results in reduced affinity to the complexedpharmaceutically active substance which is released and therebyactivated within the target cell.

EXAMPLES

[0027] (1) Synthesis of a peptide structure with preselected breakpoint

[0028] The peptide is synthesized according the well knownFmoc-procedure described by M & A Bodanzky in The Practice ofPeptideSynthesis (Springer Verlag, Heidelberg, 1984). For sequence (IV)preferably at first Fmoc-Lys(Boc)-OH is fixed on the WANG resin.Afterwards the Fmoc group is split by DMF-Piperidine and, after washingwith the sequence DMF-dichloromethane-ethanol-propanol (2), thefollowing amino acids are added as Fmoc-Asp(OtBut)-OH (alternative:Fmoc-Pro-OH), Fmoc-Tyr(OtBut)-OH, and again Fmoc-Lys(Boc)-OH, each byactivation with diisopropyl carbodiimide in the presence ofdimethylamino pyridine and/or diisopropylethylamide. Finally,carbodiimide-activated acetyl (2)-CD is coupled to the peptide and thecomplete structure is removed from the resin by trifluoroacetic acid.Afterwards, biotinylation is performed with biotin hydroxysuccininimidylester and the (2N)aminopropylamido-CD is coupled to the activatedpeptide. Preparative thin layer chromatography {Silica gel 60, solventacetic acid-acetone-methanol-benzene 5:5:20:70 (all by vol.)} is usedfor isolation with UV or iodine vapor detection, additionally sprayingwith dimethylamino cinnamic aldehyde or ninhydrin. MALDI serves forMr-determination and as a purity check. .This procedure is applicablewithout any change to all peptide structures mentioned.

[0029] (2) Test on intactness in blood samples (to be performed later onevery patient blood)

[0030] 0.1-0.5 mg of the peptide (complete sequence or sequenceaccording to (II)) are admixed to 1 ml of blood serum and incubated at37° C. for 1-8 hours. Afterwards, the sample is precipitated withperchloric acid, the supernatant neutralized with KOH, andchromatographed analytically as mentioned in example (1). The peptidespot should be unchanged under these conditions, as we found in 22patient samples.

[0031] (3) Inclusion of Paclitaxel or other cytostatic antibiotics incyclodextrin oligomers according to example (1)

[0032] 10 μM of the length-adequate dimer(according example (1)) inwatery buffer (HEPES or phosphate pH 7.4) and 10 μM Paclitaxel [or othercytostatics like colchicine, vincristine, vinblastine as given in Moser(Ser. No. 09/554,223), preferably in DMSO solution, are incubatedovernight at 50° C. Monomerization of the pharmaceutically activesubstances is followed by fluorimetry. Mitotic activity in cell culturesis documented at 40 nM pharmaceutically active substance concentrationin the presence and absence of the cyclodextrin oligomer. This solutioncan be equally prepared and used for treatment of patients as anisotonic infusion.

[0033] (4) Conditioning of tumors in patients and application of thesolution according to example (3)

[0034] According to K Bosslet et al. (Die gelben Hefte 32 149-155(1992)), a tumor in a patient is first labelled

[0035] (a) by infusion of a tumor-specific orneovascularization-specific humanized biotinylated antibody or antibodyfragment or

[0036] (b) a humanized antibody-chymotrypsin conjugate.

[0037] If in case (a) most of the antibody is eliminated from blood (aschecked e.g. from biotin content of blood samples), NeutrAvidin isinfused at adequate dosage to eliminate remnants of free antibody fromthe blood and other storage organs (Liver, kidney) and in order to labelantibody bound to the tumor. As a 3rd infusion after about one hour upto 2 liters containing up to 20 μM pharmaceutically active substance/perday according to example (III) are infused to the patient.

[0038] Having described the preferred embodiments of the invention, itis to be understood that the present invention is not limited to theseprecise embodiments, and that various changes and modifications may beeffected therein by one skilled in the art without departing from thescope or spirit of the invention as defined in the appended claims.

What is claimed is:
 1. A rigidly spaced, cyclodextrin dimer to transportpharmaceutically active substances having potential high side effectscomprising: a spacer sequence to rigidly space the cyclodextrin ends;said spacer sequence is a peptide structure; and wherein said peptidestructure is stable during transport in blood.
 2. A rigidly spaced,cyclodextrin dimer according to claim 1, wherein said peptide structurehas a preselected break point which is cleavable by intracellularenzymes but not by enzymes in blood.
 3. A rigidly spaced, cyclodextrindimer according to claim 2, wherein said breaking point within saidpeptide structure consists essentially of the sequence N-Tyr-Asp-C,which is sensitive to chymotrypsin.
 4. A rigidly spaced, cyclodextrindimer according to claim 1, wherein said peptide structure containsLys(biotinyl) for coupling said spacer sequence to an avidin-biotinsystem.
 5. A rigidly spaced, cyclodextrin dimer according to claim 1,which is complexed with a pharmaceutically active substance.
 6. Arigidly spaced, cyclodextrin dimer according to claim 1, wherein saidpeptide structure contains repeated Lys(biotinyl) moiety in adjacent orspacer configuration, providing the possibility to concentrate saidmoiety by accumulation.
 7. A rigidly spaced, cyclodextrin dimeraccording to claim 5, wherein said peptide structure contains repeatedLys(biotinyl) moiety in adjacent or spacer configuration, providing thepossibility to concentrate the complex by accumulation.
 8. A rigidlyspaced, cyclodextrin dimer according to claim 5, wherein said complexedmoiety are used in tumor therapy.
 9. A medicament containing acyclodextrin moiety according to claim 1 or a complex of saidcyclodextrin moiety with a pharmaceutically active substance.